Epigenetic dysregulation appears to be a nearly universal feature of human cancers. These epigenetic alterations can cooperate with genetic alterations to alter gene expression programs and allow cancer cells to evade the rules that typically govern normal cells. A predominant mechanism responsible for epigenetic dysregulation occurs through DNA hypermethylation at CpG dinucleotides at transcriptional regulatory regions. Such DNA hypermethylation can result in epigenetic gene repression and is thought to be mediated by 5- meCpG-binding domain (MBD) family proteins, such as MBD2, acting in multi-protein complexes containing histone modifying enzymes to direct the assembly of repressive chromatin. Targeting of epigenetic mechanisms is emerging as a promising avenue of anticancer therapy. MBD2 has been credentialed as a key mediator of DNA hypermethylation induced epigenetic gene repression, and also as a target for cancer therapy/prevention. This proposal aims to elucidate the mechanism of action of novel hit small molecules capable of inhibiting the binding of the MBD2 epigenetic reader protein to methylated CpG DNA. Cell and target-based screens have identified 20 hit compounds capable of inhibiting MBD2 binding to methylated DNA using three distinct mechanisms. These mechanisms of inhibition will be thoroughly elucidated using biochemical and biophysical techniques including time-resolved fluorescence resonance energy transfer (TR- FRET), isothermal titration calorimetry (ITC), bio-layer interferometry (BLI), and a SYBR green DNA intercalation assay. Hit compounds will be optimized for selectivity and potency through methodical structure activity relationship elucidation using the same biochemical and biophysical techniques. The two most selective and potent inhibitors will then be compared to genetic disruption of MBD2 for their ability to de- repress genes typically silenced via the MBD2-Mi2/NuRD complex in normal and cancer cell lines. MBD2 will be genetically disrupted in normal and cancer cell lines using both shRNA targeted to MBD2 in a lentiviral construct and using the CRISPR-Cas9 system with guide RNAs targeting Exon 1 of the MBD2 gene. Gene de- repression will be assessed for specific genes using quantitative real time PCR and genome wide using ChIP- seq and RNA-seq. Lead compounds identified will also be assessed for their potential as cancer preventative and therapeutic agents using clonogenic survival and the IncuCyte microscopic proliferation kinetics system in vitro and using the APC Min and xenograft mouse models in vivo. My long-term goal is to understand the role of MBD2 in epigenetic gene repression and in cancer initiation and progression. The objective here is to develop novel pharmacological probes and optimized lead inhibitors of MBD2 and test their potential for relief of DNA methylation mediated epigenetic gene repression and as cancer therapeutic agents.